Suh D Y, Kagami J, Fukuma K, Sankawa U
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, 930-0194, Japan.
Biochem Biophys Res Commun. 2000 Sep 7;275(3):725-30. doi: 10.1006/bbrc.2000.3368.
Chalcone and stilbene synthases (CHS and STS) catalyze condensation reactions of p-coumaroyl-CoA and three C(2)-units from malonyl-CoA, but catalyze different cyclization reactions to produce naringenin chalcone and resveratrol, respectively. Condensing activities of wild-type CHS and STS as well as STS-C60S mutant were inhibited by iodoacetamide (Idm) and diethyl pyrophosphate (DPC). DPC also inhibited malonyl-CoA decarboxylation activity of wild-type and C164S mutants of CHS and STS. Meanwhile, Idm treatment enhanced (two- to fourfold) malonyl decarboxylase activity of wild-type enzymes and STS-C60S, whereas this priming effect was not observed with C164S mutants of CHS and STS, indicating that the cysteine residue being modified by Idm is the catalytic Cys164 of CHS and STS. DPC inhibition of decarboxylation activity of wild-type CHS was pH-independent in the range of pH 5.8 to 7.8; however, its inhibitory effect on CHS-C164S increased as pH increased from 6.2 to 7.4 with a midpoint of 6.4. Based on the 3-D structure of CHS and the observed shift in microscopic pK(a), it was concluded that the histidine residue being modified by DPC in CHS is likely the catalytic His303 and that His303 forms an ionic pair (catalytic dyad) with Cys164 in wild-type CHS. In addition, our results showed that Cys60 in STS is not essential for the activity and only a single cysteine (Cys164) participates in the catalysis as in CHS.
查尔酮合酶和芪合酶（CHS和STS）催化对香豆酰辅酶A与丙二酰辅酶A的三个C(2)单元的缩合反应，但分别催化不同的环化反应生成柚皮素查尔酮和白藜芦醇。野生型CHS和STS以及STS - C60S突变体的缩合活性受到碘乙酰胺（Idm）和焦磷酸二乙酯（DPC）的抑制。DPC还抑制了CHS和STS野生型及C164S突变体的丙二酰辅酶A脱羧活性。同时，Idm处理增强了（两到四倍）野生型酶和STS - C60S的丙二酰脱羧酶活性，而CHS和STS的C164S突变体未观察到这种引发效应，表明被Idm修饰的半胱氨酸残基是CHS和STS的催化性Cys164。在pH 5.8至7.8范围内，DPC对野生型CHS脱羧活性的抑制与pH无关；然而，随着pH从6.2增加到7.4，其对CHS - C164S的抑制作用增强，中点为6.4。基于CHS的三维结构和观察到的微观pK(a)变化，得出结论：CHS中被DPC修饰的组氨酸残基可能是催化性His303，并且在野生型CHS中His303与Cys164形成离子对（催化二元组）。此外，我们的结果表明，STS中的Cys60对活性不是必需的，并且与CHS一样，只有单个半胱氨酸（Cys164）参与催化。
